RESUMEN
YgjD from COG0533 is amongst a small group of highly conserved proteins present in all three domains of life. Various roles and biochemical functions (including sialoprotease and endonuclease activities) have been ascribed to YgjD and orthologs, the most recent, however, is involvement in the post transcriptional modification of certain tRNAs by formation of N6-threonyl-adenosine (t6A) at position 37. In bacteria, YgjD is essential and along with YeaZ, YjeE, and YrdC has been shown to be 'necessary and sufficient' for the tRNA modification. To further define interactions and possible roles for some of this set of proteins we have undertaken structural and biochemical studies. We show that formation of the previously reported heterodimer of YgjD-YeaZ involves ordering of the C-terminal region of YeaZ which extends along the surface of YgjD in the crystal structure. ATPγS or AMP is observed in YgjD while no nucleotide is bound on YeaZ. ITC experiments reveal previously unreported binary and ternary complexes which can be nucleotide dependent. The stoichiometry of the YeaZ-YgjD complex is 1:1 with a K(D) of 0.3 µM. YgjD and YjeE interact only in the presence of ATP, while YjeE binds to YgjD-YeaZ in the presence of ATP or ADP with a K(D) of 6 µM. YgjD doesn't bind the precursors of t6A, threonine, and bicarbonate. These results show a more complex set of interactions than previously thought, which may have a regulatory role. The understanding gained should help in deriving inhibitors of these essential proteins that might have potential as antibacterial drugs.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/química , Salmonella typhimurium/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Calorimetría , Cristalografía por Rayos X , Humanos , Nucleótidos/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas , Multimerización de Proteína , ARN de Transferencia/metabolismoRESUMEN
Respiratory syncytial virus (RSV) is a frequent cause of respiratory illness in infants, but there is currently no vaccine nor effective drug treatment against this virus. The RSV RNA genome is encapsidated and protected by a nucleocapsid protein; this RNA-nucleocapsid complex serves as a template for viral replication. Interest in the nucleocapsid protein has increased owing to its recent identification as the target site for novel anti-RSV compounds. The crystal structure of human respiratory syncytial virus nucleocapsid (HRSVN) was determined to 3.6 Å resolution from two crystal forms belonging to space groups P2(1)2(1)2(1) and P1, with one and four decameric rings per asymmetric unit, respectively. In contrast to a previous structure of HRSVN, the addition of phosphoprotein was not required to obtain diffraction-quality crystals. The HRSVN structures reported here, although similar to the recently published structure, present different molecular packing which may have some biological implications. The positions of the monomers are slightly shifted in the decamer, confirming the adaptability of the ring structure. The details of the inter-ring contacts in one crystal form revealed here suggest a basis for helical packing and that the stabilization of native HRSVN is via mainly ionic interactions.
Asunto(s)
Proteínas de la Nucleocápside/química , Virus Sincitial Respiratorio Humano/química , Cristalografía por Rayos X , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , ARN Viral/químicaRESUMEN
Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and approximately 5 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 A, and space group P2(1), with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 A, beta = 90.1 degrees , respectively. For unlabelled HRSVN, only crystals belonging to space group P2(1) were obtained that diffracted to 3.6 A. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.
Asunto(s)
Proteínas de la Nucleocápside/química , Virus Sincitial Respiratorio Humano/química , Cristalización , Humanos , Lactante , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Virus Sincitial Respiratorio Humano/genética , Difracción de Rayos XRESUMEN
The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.
Asunto(s)
GTP Fosfohidrolasas/química , Salmonella typhimurium/enzimología , Secuencia de Aminoácidos , Calorimetría , Cristalización , Cristalografía por Rayos X , GTP Fosfohidrolasas/metabolismo , Datos de Secuencia Molecular , Alineación de Secuencia , TermodinámicaRESUMEN
The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.
Asunto(s)
Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Salmonella typhimurium/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clonación Molecular , Biología Computacional , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Biblioteca de Péptidos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Dehydroquinate synthase (DHQS) is a potential target for the development of novel broad-spectrum antimicrobial drugs, active against both prokaryotes and lower eukaryotes. Structures have been reported for Aspergillus nidulans DHQS (AnDHQS) in complexes with a range of ligands. Analysis of these AnDHQS structures showed that a large-scale domain movement occurs during the normal catalytic cycle, with a complex series of structural elements propagating substrate binding-induced conformational changes away from the active site to distal locations. Compared to corresponding fungal enzymes, DHQS from bacterial species are both mono-functional and significantly smaller. We have therefore determined the structure of Staphylococcus aureus DHQS (SaDHQS) in five liganded states, allowing comparison of ligand-induced conformational changes and mechanisms of domain closure between fungal and bacterial enzymes. This comparative analysis shows that substrate binding initiates a large-scale domain closure in both species' DHQS and that the active site stereochemistry, of the catalytically competent closed-form enzyme thus produced, is also highly conserved. However, comparison of AnDHQS and SaDHQS open-form structures, and analysis of the putative dynamic processes by which the transition to the closed-form states are made, shows a far lower degree of similarity, indicating a significant structural divergence. As a result, both the nature of the propagation of conformational change and the mechanical systems involved in this propagation are quite different between the DHQSs from the two species.
Asunto(s)
Células Eucariotas/enzimología , Liasas de Fósforo-Oxígeno/química , Células Procariotas/enzimología , Conformación Proteica , Secuencia de Aminoácidos , Aspergillus nidulans/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligandos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , Organofosfonatos/química , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Alineación de Secuencia , Staphylococcus aureus/enzimologíaRESUMEN
Two high-resolution structures have been determined for Eschericia coli aspartate beta-semialdehyde dehydrogenase (ecASADH), an enzyme of the aspartate biosynthetic pathway, which is a potential target for novel antimicrobial drugs. Both ASADH structures were of the open form and were refined to 1.95 A and 1.6 A resolution, allowing a more detailed comparison with the closed form of the enzyme than previously possible. A more complex scheme for domain closure is apparent with the subunit being split into two further sub-domains with relative motions about three hinge axes. Analysis of hinge data and torsion-angle difference plots is combined to allow the proposal of a detailed structural mechanism for ecASADH domain closure. Additionally, asymmetric distortions of individual subunits are identified, which form the basis for the previously reported "half-of-the-sites reactivity" (HOSR). A putative explanation of this arrangement is also presented, suggesting the HOSR system may provide a means for ecASADH to offset the energy required to remobilise flexible loops at the end of the reaction cycle, and hence avoid falling into an energy minimum.
Asunto(s)
Aspartato-Semialdehído Deshidrogenasa/química , Escherichia coli/enzimología , Sitios de Unión , Cristalografía por Rayos X , Bases de Datos como Asunto , Dimerización , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaAsunto(s)
Hidroliasas/química , Ácido Quínico/análogos & derivados , Staphylococcus aureus/enzimología , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Hidroliasas/metabolismo , Ligandos , Modelos Moleculares , Unión Proteica , Ácido Quínico/metabolismo , Salmonella typhi/enzimología , Homología Estructural de ProteínaRESUMEN
Crystallization of Aspergillus nidulans 3-dehydroquinate synthase (DHQS), following turnover of the enzyme by addition of the substrate DAHP, gave a new crystal form (form J). Although the crystals have dimensions of only 50 x 20 x 5 micro m, they are well ordered, diffracting to 1.7 A. The space group is C222(1), with unit-cell parameters a = 90.0, b = 103.7, c = 177.4 A. Structure determination and refinement to R = 0.19 (R(free) = 0.25) shows the DHQS is in the 'open' form with the substrate site unoccupied but with some loop regions perturbed. Previous crystals of open-form DHQS only diffracted to 2.5 A resolution. The use of enzyme turnover may be applicable in other systems in attempts to improve crystal quality.
Asunto(s)
Aspergillus nidulans/enzimología , Liasas de Fósforo-Oxígeno/química , Bioquímica/métodos , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Liasas de Fósforo-Oxígeno/metabolismo , Conformación Proteica , Homología Estructural de Proteína , Azúcares Ácidos/química , Azúcares Ácidos/metabolismoRESUMEN
Leu100Ile, Val106Ala and Val108Ile are mutations in HIV-1 reverse transcriptase (RT) that are observed in the clinic and give rise to resistance to certain non-nucleoside inhibitors (NNRTIs) including the first-generation drug nevirapine. In order to investigate structural mechanisms of resistance for different NNRTI classes we have determined six crystal structures of mutant RT-inhibitor complexes. Val108 does not have direct contact with nevirapine in wild-type RT and in the RT(Val108Ile) complex the biggest change observed is at the distally positioned Tyr181 which is > 8 A from the mutation site. Thus in contrast to most NNRTI resistance mutations RT(Val108Ile) appears to act via an indirect mechanism which in this case is through alterations of the ring stacking interactions of the drug particularly with Tyr181. Shifts in side-chain and inhibitor positions compared to wild-type RT are observed in complexes of nevirapine and the second-generation NNRTI UC-781 with RT(Leu100Ile) and RT(Val106Ala), leading to perturbations in inhibitor contacts with Tyr181 and Tyr188. Such perturbations are likely to be a factor contributing to the greater loss of binding for nevirapine compared to UC-781 as, in the former case, a larger proportion of binding energy is derived from aromatic ring stacking of the inhibitor with the tyrosine side-chains. The differing resistance profiles of first and second generation NNRTIs for other drug resistance mutations in RT may also be in part due to this indirect mechanism.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Codón , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , Mutación , Nevirapina/metabolismo , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
In order to investigate systematically substrate and cofactor-induced conformational changes in the enzyme dehydroquinate synthase (DHQS), eight structures representing a series of differently liganded states have been determined in a total of six crystal forms. DHQS in the absence of the substrate analogue carbaphosphonate, either unliganded or in the presence of NAD or ADP, is in an open form where a relative rotation of 11-13 degrees between N and C-terminal domains occurs. Analysis of torsion angle difference plots between sets of structures reveals eight rearrangements that appear relevant to domain closure and a further six related to crystal packing. Overlapping 21 different copies of the individual N and C-terminal DHQS domains further reveals a series of pivot points about which these movements occur and illustrates the way in which widely separated secondary structure elements are mechanically inter-linked to form "composite elements", which propagate structural changes across large distances. This analysis has provided insight into the basis of DHQS ligand-initiated domain closure and gives rise to the proposal of an ordered sequence of events involving substrate binding, and local rearrangements within the active site that are propagated to the hinge regions, leading to closure of the active-site cleft.
Asunto(s)
Adenosina Difosfato/farmacología , Aspergillus nidulans/enzimología , NAD/farmacología , Organofosfonatos/farmacología , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Solventes , Electricidad Estática , Especificidad por SustratoRESUMEN
The HIV-2 serotype of HIV is a cause of disease in parts of the West African population, and there is evidence for its spread to Europe and Asia. HIV-2 reverse transcriptase (RT) demonstrates an intrinsic resistance to non-nucleoside RT inhibitors (NNRTIs), one of two classes of anti-AIDS drugs that target the viral RT. We report the crystal structure of HIV-2 RT to 2.35 A resolution, which reveals molecular details of the resistance to NNRTIs. HIV-2 RT has a similar overall fold to HIV-1 RT but has structural differences within the "NNRTI pocket" at both conserved and nonconserved residues. The structure points to the role of sequence differences that can give rise to unfavorable inhibitor contacts or destabilization of part of the binding pocket at positions 101, 106, 138, 181, 188, and 190. We also present evidence that the conformation of Ile-181 compared with the HIV-1 Tyr-181 could be a significant contributory factor to this inherent drug resistance of HIV-2 to NNRTIs. The availability of a refined structure of HIV-2 RT will provide a stimulus for the structure-based design of novel non-nucleoside inhibitors that could be used against HIV-2 infection.
Asunto(s)
ADN Polimerasa Dirigida por ARN/química , Fármacos Anti-VIH/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Farmacorresistencia Viral , Transcriptasa Inversa del VIH , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-ActividadRESUMEN
Six structures of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing combinations of resistance mutations for zidovudine (AZT) (M41L and T215Y) or lamivudine (M184V) have been determined as inhibitor complexes. Minimal conformational changes in the polymerase or nonnucleoside RT inhibitor sites compared to the mutant RTMC (D67N, K70R, T215F, and K219N) are observed, indicating that such changes may occur only with certain combinations of mutations. Model building M41L and T215Y into HIV-1 RT-DNA and docking in ATP that is utilized in the pyrophosphorolysis reaction for AZT resistance indicates that some conformational rearrangement appears necessary in RT for ATP to interact simultaneously with the M41L and T215Y mutations.
Asunto(s)
Fármacos Anti-VIH/farmacología , Codón , Farmacorresistencia Viral/genética , Transcriptasa Inversa del VIH/química , Lamivudine/farmacología , Mutación , Zidovudina/farmacología , Adenosina Trifosfato/metabolismo , Cristalización , Transcriptasa Inversa del VIH/genética , Conformación ProteicaRESUMEN
NmrA is a negative transcriptional regulator involved in the post-translational modulation of the GATA-type transcription factor AreA, forming part of a system controlling nitrogen metabolite repression in various fungi. X-ray structures of two NmrA crystal forms, both to 1.8 A resolution, show NmrA consists of two domains, including a Rossmann fold. NmrA shows an unexpected similarity to the short-chain dehydrogenase/reductase (SDR) family, with the closest relationship to UDP-galactose 4-epimerase. We show that NAD binds to NmrA, a previously unreported nucleotide binding property for this protein. NmrA is unlikely to be an active dehydrogenase, however, as the conserved catalytic tyrosine in SDRs is absent in NmrA, and thus the nucleotide binding to NmrA could have a regulatory function. Our results suggest that other transcription factors possess the SDR fold with functions including RNA binding. The SDR fold appears to have been adapted for other roles including non-enzymatic control functions such as transcriptional regulation and is likely to be more widespread than previously recognized.
Asunto(s)
Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transcripción Genética , UDPglucosa 4-Epimerasa/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Electrones , Proteínas Fúngicas/química , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Neurospora crassa/enzimología , Unión Proteica , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Tirosina/metabolismoRESUMEN
The NmrA repressor protein of Aspergillus nidulans was overproduced in Escherichia coli and purified to homogeneity. Gel-exclusion chromatography showed that NmrA was monomeric in solution under the buffer conditions used. The protein was crystallized in three forms, belonging to trigonal, monoclinic and hexagonal space groups. Two of these crystal forms (A and B) diffract to high resolution and thus appear suitable for structure determination. Crystal form A belongs to space group P3((1))21, with unit-cell parameters a = b = 76.8, c = 104.9 A. Crystal form B belongs to space group C2, with unit-cell parameters a = 148.8, b = 64.3, c = 110.2 A, beta = 121.8 degrees.
Asunto(s)
Aspergillus nidulans/química , Proteínas Fúngicas/química , Proteínas Represoras/química , Cristalización , Cristalografía por Rayos X , Escherichia coli , Compuestos de Nitrógeno , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Mutations at either Tyr181 or Tyr188 within HIV-1 reverse transcriptase (RT) give high level resistance to many first generation non-nucleoside inhibitors (NNRTIs) such as the anti-AIDS drug nevirapine. By comparison second generation inhibitors, for instance the drug efavirenz, show much greater resilience to these mutations. In order to understand the structural basis for these differences we have determined a series of seven crystal structures of mutant RTs in complexes with first and second generation NNRTIs as well as one example of an unliganded mutant RT. These are Tyr181Cys RT (TNK-651) to 2.4 A, Tyr181Cys RT (efavirenz) to 2.6 A, Tyr181Cys RT (nevirapine) to 3.0 A, Tyr181Cys RT (PETT-2) to 3.0 A, Tyr188Cys RT (nevirapine) to 2.6 A, Tyr188Cys RT (UC-781) to 2.6 A and Tyr188Cys RT (unliganded) to 2.8 A resolution. In the two previously published structures of HIV-1 reverse transcriptase with mutations at 181 or 188 no side-chain electron density was observed within the p66 subunit (which contains the inhibitor binding pocket) for the mutated residues. In contrast the mutated side-chains can be seen in the NNRTI pocket for all seven structures reported here, eliminating the possibility that disordering contributes to the mechanism of resistance. In the case of the second generation compounds efavirenz with Tyr181Cys RT and UC-781 with Tyr188Cys RT there are only small rearrangements of either inhibitor within the binding site compared to wild-type RT and also for the first generation compounds TNK-651, PETT-2 and nevirapine with Tyr181Cys RT. For nevirapine with the Tyr188Cys RT there is however a more substantial movement of the drug molecule. We conclude that protein conformational changes and rearrangements of drug molecules within the mutated sites are not general features of these particular inhibitor/mutant combinations. The main contribution to drug resistance for Tyr181Cys and Tyr188Cys RT mutations is the loss of aromatic ring stacking interactions for first generation compounds, providing a simple explanation for the resilience of second generation NNRTIs, as such interactions make much less significant contribution to their binding.
Asunto(s)
Codón/genética , Farmacorresistencia Microbiana/genética , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Mutación Missense/genética , Inhibidores de la Transcriptasa Inversa/farmacología , Sitios de Unión , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
Using a large panel of human immunodeficiency virus type 1 site-directed mutants, we have observed a higher correlation than has previously been demonstrated between zidovudine (AZT)-triphosphate resistance data at the reverse transcriptase (RT) level and corresponding viral AZT resistance. This enhanced-resistance effect at the RT level was seen with ATP and to a lesser extent with PP(i) when ATP was added at physiological concentrations. The ATP-dependent mechanism (analogous to pyrophosphorolysis) appears to be dominant in the mutants bearing the D67N and K70R or 69 insertion mutations, whereas the Q151M mutation seems independent of ATP for decreased binding to AZT-triphosphate.
Asunto(s)
Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/efectos de los fármacos , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Nucleótidos de Timina/farmacología , Zidovudina/farmacología , Adenosina Trifosfato/metabolismo , Didesoxinucleótidos , Farmacorresistencia Microbiana , Guanosina Trifosfato/metabolismo , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , Mutagénesis Sitio-Dirigida , Zidovudina/análogos & derivadosRESUMEN
We have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.
Asunto(s)
Adenosina Trifosfato/metabolismo , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Estavudina/farmacología , Sustitución de Aminoácidos , Sitios de Unión , Farmacorresistencia Microbiana/fisiología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/genética , Inhibidores de la Transcriptasa Inversa/metabolismo , Estavudina/metabolismoRESUMEN
A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-activity relationship (SAR) studies suggested that a meta substituent, particularly a meta methyl substituent, invariably increased antiviral activities. However, optimal antiviral activities were manifested by compounds where both meta groups in the arylsulfonyl moiety were substituted and one of the substituents was a methyl group. Such a disubstitution led to compounds 3v, 3w, 3x, and 3y having IC50 values against HIV-1 in the low nanomolar range. When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u-z and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against the V106A and P236L strains and submicromolar to low nanomolar activity against strains E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v (739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure allowed for the rationalization of SAR data and potencies of the compounds against the mutants.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Nitrilos/síntesis química , Sulfonas/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Línea Celular Transformada , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/química , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Nitrilos/química , Nitrilos/farmacología , Conformación Proteica , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
Extensive crystallization trials of Aspergillus nidulans dehydroquinate synthase, a potential novel target for antimicrobial drugs, in complexes with different ligands have resulted in the identification of nine crystal forms. Crystals of unliganded DHQS, binary complexes with either the substrate analogue, carbaphosphonate or the cofactor NADH, as well as the ternary DHQS-carbaphosphonate-cofactor complex, were obtained. The ternary complex crystallizes from ammonium sulfate and CoCl(2) in space group P2(1)2(1)2, with unit-cell parameters a = 133.8, b = 86.6, c = 74.9 A. The binary carbaphosphonate complex crystallizes from PEG 6000 in space group P2(1)2(1)2(1), with a = 70.0, b = 64.0, c = 197.6 A, and the binary cofactor complex crystallizes from PEG 3350 and sodium potassium tartrate in space group P2(1), with a = 83.7, b = 70.4, c = 144.3 A, beta = 89.2 degrees. DHQS in the absence of ligands crystallizes in space group P2(1), with a = 41.0, b = 68.9, c = 137.7 A, beta = 94.8 degrees. Each of these crystal forms are suitable for high-resolution structure determination. Structures of a range of DHQS-ligand complexes will be of value in the structure-based design of novel antimicrobial drugs.